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Advances in Radiotherapy
& Nuclear Medicine Aspirin’s protective effect on RISI

3.2. Main cell type annotation and response to IFE B was further differentiated into IFE B1 and IFE
irradiation B2, IFE D was differentiated into IFE D1 and IFE D2,
Analysis of cell populations in irradiated versus control and the uHF was differentiated into uHFII, uHFIV, and
groups revealed notable differences in the composition of uHFVI cells, resulting in a total of 13 annotated subtypes
major skin cell types. A total of 9,022 cells were classified (Figure S1D-G, Figure 3B).
into major cell types, including epithelial cells, fibroblasts, The t-SNE plot visualization (Figure 3A) clearly
T cells, monocytes, and endothelial cells, visualized using shows the separation between these 13 subtypes, with
t-SNE. Epithelial cells constituted the largest population, clusters highlighted for each cell population. A bar plot
followed by fibroblasts, T cells, and other types (Figure 2A). representation of cell numbers (Figure 3C) revealed
Comparing control and irradiated groups, the average variations in cell distributions between the control
number of cells within each type showed significant changes, and irradiated groups, while the cell proportion plot
with a marked reduction in the number of epithelial cells (Figure 3D) depicted distinct changes in the prevalence of
in the irradiated group (Figure 2B). The proportion of each subtype under irradiation conditions. Notably, despite
cell types also varied, suggesting that radiation exposure the overall reduction in cell number post-irradiation, the
disproportionately affected specific cell populations, number and proportion of IFE B1 cells were increased in
reducing epithelial content while maintaining more stable the irradiated group compared to the control group. As
levels of T cells and monocytes. Marker gene analysis this is an intriguing observation, subsequent analyses were
showed differential expression across cell types, with key primarily focused on the IFE B cell population.
markers such as Epcam, Cd44, Ly6c2, and Pecam1 being
particularly notable. For example, Ly6c2, which is related Further analysis examined the cell cycle distribution of
to immune response, displayed elevated levels in irradiated the two IFE B cell subtypes and revealed that the majority
samples, indicating an increased presence of monocytes in of cells in the G2M phase were present in the IFE B1
response to radiation-induced inflammation (Figure 2C). subpopulation (Figure 3E and F). Compared to other IFE
The t-SNE visualizations focusing on the expression of B cells, IFE B1 cells exhibited higher expression levels of
key marker genes across different cell types demonstrated Cdk1 and Ccnb1, leading us to define these actively cycling
significant radiation-induced changes. Epithelial cells and cells as IFE Cycling (IFE C) cells (Figure 3G and H).
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fibroblasts exhibited an upregulation of genes associated We then analyzed stem cell marker expression across
with extracellular matrix remodeling, reflecting an attempt different IFE cell subtypes, revealing that IFE C cells
to repair radiation damage, potentially contributing exhibited the highest expression of the stem cell marker
to fibrosis as part of the skin’s response (Figure 2D). Krt14, suggesting the highest level of stemness within this
Pathway analysis of DEGs across cell types highlighted the subgroup (Figure 3I). In addition, comparing control and
involvement of multiple key-signaling pathways, including irradiated groups, we found that the expression of cell cycle
“cornified envelope,” “extracellular matrix,” “chemokines,” markers Cdk1 and Ccnb1 was reduced post-irradiation,
“fatty acyl-CoA synthesis,” and “Rho GTPase” pathways. whereas the expression of the stemness marker Krt14
Notably, the “cornified envelope” and “extracellular matrix” was elevated in the irradiated group (Figure 3J). Based on
pathways were significantly upregulated in irradiated these observations, we hypothesize that IFE C cells serve
epithelial cells, suggesting disruptions in skin barrier as a reservoir for IFE cell populations. Upon irradiation,
function and increased extracellular matrix activity – key IFE C cells in mouse skin experience cell cycle arrest in
features of the response to radiation injury (Figure 2E). the G2M phase. However, should these cells successfully
bypass the G2M checkpoint, they could rapidly proliferate
3.3. Cell subtype annotation analysis and differentiate to replenish epidermal cells in the skin.
The analysis of cell subtypes identified 13 distinct cell 3.4. Pseudotime analysis of IFE cells
populations. This process was conducted in two major
steps. Initially, using known skin cell markers, eight A pseudotime analysis was performed on the major
primary cell types were annotated, including IFE B, IFE cell populations to understand their differentiation
IFE differentiated (IFE D), IFE K, infundibulum basal trajectories. Figure 4 (A-C) shows the differentiation path
(INFU B), outer bulge (OB), inner bulge (IB), SG, and of IFE subtypes, with IFE C cells positioned upstream
LH (Figure S1A-C). In the next step, four of these major in the differentiation process, confirming our previous
cell types underwent further characterization through hypothesis that IFE C serves as an origin cell type for further
comprehensive multigene expression analysis. This differentiation. The trajectory visualization highlights that
additional analysis allowed for finer differentiation of these IFE C cells are found at the beginning of the differentiation
groups, leading to the identification of specific subtypes : path, with subsequent transitions to other subtypes.
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Volume 3 Issue 1 (2025) 62 doi: 10.36922/arnm.5829